E-selectin, a cytokine-inducible, endothelial-specific member of the Selectin family of adhesion receptors, supports the rolling and transition to stable arrest of selected blood leukocytes on the surface of activated vascular endothelium in vitro and in vivo. In this project we will test the hypothesis that E-selectin, in addition to its adhesive functions, mediates intercellular communication between activated endothelium and transiting leukocytes, and thus plays an active role in the regulation of endothelial cell adaptive responses during leukocyte-endothelial interactions at sites of inflammation. In Specific Aim #1, the early paracrine and autocrine endothelial cell signaling responses, elicited by leukocyte tethering via E-selectin will be assessed by: (i) defining the production of bioactive molecules (in particular, nitric oxide) after E-selectin ligation; (ii) characterizing the endothelial cytoskeletal and lateral junctional remodeling associated with ligation of E-selectin, using a combination of biochemistry and live-time molecular imaging; and (iii) determining the functional implications of these endothelial responses, using quantitative in vitro assays of leukocyte activation, adhesion and transendothelial migration. In Specific #2, (i) the transcriptional programs evoked after ligation of E-selectin at the endothelial cell surface will be dissected, using oligonucleotide-based microarrays and computational biology tools; (ii) the role of specific signaling motifs in the cytoplasmic domain of E-selectin in evoking distinct gene expression programs will be determined; and (iii) the resultant modulated endothelial phenotype will be characterized using transcriptional programs as descriptors/predictors of cellular functions and processes. In Specific Aim #3. (i) the integrative pathophysiological implications of these patterns of E-selectin-mediated endothelial phenotypic modulation will be critically examined, initially in vitro, using cultured endothelial cells derived from wild-type and E-selectin-null mice, expressing normal and adenovirally transduced mutant E-selectin molecules; and then, (ii) selected in vivo models of inflammation will be used to critically assess the pathophysiological relevance of these processes in wild-type and E-selectin-null mice.